Antibodies are immunological proteins that bind specifically to antigens. At present, many monoclonal antibodies have been developed as anti-cancer agents or are used in cancer therapy. However, almost all antibodies, although inhibitory of the growth of cancer cells and thus suppressive of the progression of cancer, have very limited application in the therapy of cancers [Sharkey et al., Adv. Drug Del. Rev., 2008, 1407-1420]. As an alternative to antibody therapeutic agents to overcome this limitation, an antibody-drug conjugate (ADC), in which an antibody is coupled with a drug, has arisen [Chari, Acc. Chem. Res., 41(2008), 98-107]. As used herein, the term “ADC” refers to a highly potent biopharmaceutical drug that comprises a cytotoxic drug or toxin conjugated with a monoclonal antibody (mAb) capable of allowing the cytotoxic drug or toxin to be selectively delivered into a target tumor cell (internalization). Given to patients, ADCs bind to target cells via the antibody moiety and are internalized into the cells, wherein the cytotoxic drug or toxin moiety is separated from the conjugate and exerts its therapeutic efficacy [Hamblett et al., Clin. Cancer Res., 10:7063-7070, Oct. 15, 2004].
For the preparation of ADCs, a suitable form of linker is usually used to couple an antibody with a cytotoxic drug. Representative among the linkers are hydrazones, disulfides, and peptides. Fundamentally, the effective performance of an ADC requires at least a certain functional level of all three components: mAb, linker, and cytotoxic drug [Chari, Acc. Chem. Res., 41(2008), 98-107].
U.S. Pat. No. 6,214,345 discloses an ADC in which a cytotoxic drug, such as doxorubicin, Taxol, etc., is coupled to an antibody via an enzyme-cleavable peptide linker that can be cleaved by the lysosomal protease cathepsin. Also, U.S. Pat. No. 7, 964,566 discloses an ADC in which a cytotoxic drug, such as MeVal-Val-Dil-Dap-norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), is attached to an antibody through an enzyme-cleavable peptide linker.
According to conventional preparation methods of ADCs using enzyme-cleavable peptide linkers, attachment between linkers and antibodies is achieved by reducing a disulfide bond in antibodies to a thiol group in the presence of a reducing agent such as dithiothreitol (DTT), and bonding the linkers to the thiol group of the antibodies. However, cleavage of an intracellular disulfide bond of an antibody by use of a reducing agent necessarily causes a structural modification of the antibody itself, which may result in a negative influence on the affinity of the antibody to its antigen. Also, such reduction may cause the antibody to undergo partial degradation, making it difficult to purify the final ADC. Further, when reduced from the intramolecular disulfide bond to a thiol group, the antibodies readily aggregate together, lowering the final production yield of ADC.
Instead of the reduction of intramolecular disulfide bonds of antibodies to thiol groups, thiolation may be achieved by reacting a reagent for sulfhydryl addition, such as 2-iminotholane (Traut's reagent), with a lysine residue of an antibody. In this case, the number of the thiolation reagent attached to an antibody shows a wide distribution ranging from zero to less than ten. Thus, the linker cannot cover all the produced thiol groups, resulting in co-existence of coupled and uncoupled thiol groups in the same antibody. Due to the thiol groups that remain uncoupled with the linker and are thus unnecessary, and residues from the thiolation reagent, the ADC prepared by this conventional method is therefore poor in performance, particularly, in pharmacokinetic properties in vivo.